Bacteria of the Acidithiobacillus genus are acidophilic, autotrophous and chemolithotrophs, in other words, they live with acid pHs of 0 to 4, their source of carbon is CO2, and their source of energy is inorganic compounds. Two of the species of this genus are of vital importance in biomining: Acidithiobacillus ferrooxidans, and Acidithiobacillus thiooxidans On the other hand, Acidithiobacillus caldus is becoming increasingly important in biomining processes.
Biomining is, generally speaking, the use of microorganisms for extracting metals from ores. Its most traditional and important expression is bioleaching, but biomining is more than this process alone, it is also the monitoring and intervention of the microorganisms involved—insofar as these techniques are complex and constantly developing—as well as laboratory-level research associated with the improvement of processes or the development of new technologies.
Bioleaching is defined as method for solubilizing metals from complex matrixes in an acid medium, employing the direct or indirect action of microorganisms (Rawlings D. E. Microb. Cell Fact. 2005; 4(1):13). It is direct when the microorganisms act on the metal or on its counter ion, releasing an ion of the metal of interest into the solution in both cases. On the other hand, it is indirect when the microorganism does not have either the metal of interest or its counter ion as a substrate, but generates chemical conditions that accelerate and favour solubilization of the said metal, whether by acidification of the medium, (for example, generating sulphuric acid) or because it generates an oxidizing agent that finally interacts with the salt (metal and counter ion) that needs to be solubilized. Species belonging to the Acidithiobacillus genus, both Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, and Acidithiobacillus caldus, are capable of producing elements that increase the oxidizing velocity of reduced sulfur compounds (such as sulfide, elemental sulfur, thionates, etc.) using oxygen as an electronic acceptor. During this process, they generate sulfuric acid as an end product, and reducing-species such as sulfite and thiosulfate, as intermediate products, which makes solubilizing the metals associated to sulfides in the ore possible particularly speaking, Acidithiobacillus ferrooxidans contributes with biological components that favor the oxidization of iron (II) to iron (III) using oxygen as an electron acceptor. The generated iron (III) is a great oxidizing agent that can oxidize the sulfides present, or any compound that needs to be oxidized.
Given the importance of bacteria of the Acidithiobacillus genus, it would be convenient to be able to genetically manipulate them—having an effective method for transforming them—whether for improving their oxidizing activity, incorporating functions of interest such as resistance to toxic compounds into them, or for getting to know more about their metabolism.
The most traditional way to modify the genetic load of bacteria is using the transformation process. This process consists in directly integrating a DNA fragment of interest into a microorganism to be transformed. There are a variety of transformation techniques in the technique, one of the most usual of which is electroporation.
All manipulations of Acidithiobacillus spp. are complex; even culturing it in the laboratory is not simple. This is because it must be kept at an extremely acid pH, and the ores it employs as a substrate are problematic, or precipitate in the medium in the form of salts, as occurs with iron, or are particulates such as elemental sulfur. The presence of iron precipitate can be observed with the naked eye as a reddish nucleus in colonies. On the other hand, bacteria adhere to particulate material, leading to a loss of biomass in the wash stages that are necessary for any microbiological manipulation technique.
Because of the above, it is necessary to be provided with an adequate transformation method especially designed for these bacteria. A method has been designed with the purpose of successfully transforming bacteria of the Acidithiobacillus genus such as the Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus species.
One of the most usual techniques used for the transformation of microorganisms, as stated above, is electroporation. Electroporation basically consists in temporarily permeabilizing the cell membrane, employing brief high-intensity electrical discharges. Transient permeable regions or structures known as micropores, through which the vectors enter the cells, are generated in the membrane.
Electroporation of Acidithiobacillus spp. becomes difficult if ferric sulfide has been used as a substrate, because the presence of ferrous ion salts associated to the bacteria could harm the transformation vectors. On the other hand, if sulfur has been used as a substrate, the cultures will be depleted after washings because part of the bacteria will be lost adhered to the particulate material.
But not only the method should be differentiated, the genetic vectors employed should have certain characteristics that allow them to be functional inside the cell. One of these corresponds to the origins of replication (ori). In some cases they are specific for different genera and even specific for one particular species, and because of this, even if we were able to transform an Acidithiobacillus spp., we would not achieve expression of the vector unless we had a replication system that were appropriate for Acidithiobacillus spp. This ideally requires that an Acidithiobacillus spp. plasmid be obtained, and its ori identified to incorporate it into the transformation vector. It is also convenient to have a promoter with a strong expression to ensure transcription of the gene of interest.
Very few Acidithiobacillus spp. plasmids have been described to date, and much less characterized. Rawlings has studied two of them (Rawlings D. E., 2005, Plasmid 53: 137-147), but has not described their use in transformations.
In the publication by English (English et al 1995, Appl. Environ. Microbiol., 61: 3256-3260), Acidithiobacillus neapolitanus is transformed by electroporation with a vector constructed with the replicon of an isolated Acidithiobacillus intermedius plasmid and with the gene of a peptide from the formation of carboxysomes in Acidithiobacillus neapolitanus. In this case, even if transformation did occur, the expression of the incorporated protein was not perceived, so it cannot be given the category of a successful transformation.
Another publication dealing with the subject of Acidithiobacillus transformation is one by Kusano (Kusano et al, 1992, J. Bacteriol., 174: 6617-6623), in which Acidithiobacillus ferrooxidans are transformed but plasmids of this species are not disclosed. In fact, the authors generated vectors with a mercury-resistant operon, a mer operon, previously isolated from Acidithiobacillus ferrooxidans. Thirty independent strains of Acidithiobacillus ferrooxidans were electroporated, and the transformation only came about in one of them, with an efficiency of 120 to 200 mercury-resistant colonies per μg of vector. This transformation efficiency is very low, so it is not considered to be an adequate transformation alternative. In fact, there are no other works by Kusano dealing with this technique, and its subsequent applicability has not been verified, because it has not been possible for other groups working with Acidithiobacillus spp. to reproduce the results of this publication.
There are three publications in which transformation of Acidithiobacillus spp. by conjugation using E. coli as a donator is achieved (Yankofsky et al, 1983, J. Bacteriol., 153: 652-657), (Jin et al 1992, Appl. Environ. Microbiol., 58: 429-430) and (Liu et al 2000, J. Bacteriol., 182: 2269-2276), but good transformation efficiency is not obtained in any of them.
Summing up, in the state of the art there are neither specific functional plasmids for the Acidithiobacillus spp. genus nor a method permitting their efficient transformation, particularly that of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus species.
This technical issue has been resolved by constructing specific functional plasmids for the Acidithiobacillus spp. genus, containing the origins of replication (ori) of isolated Acidithiobacillus ferrooxidans (Wenelen DSM 16786) and Acidithiobacillus thiooxidans (Licanantay DSM 17318) plasmids associated to an expression promoter isolated from the Wenelen (DSM 16786) strain, and by promoting and developing a specific transformation method for Acidithiobacillus spp.
Invention Abstract
The present invention discloses plasmids that allow Acidithiobacillus spp. bacteria such as the Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidithiobacillus caldus species, to be successfully transformed, and a transformation method.
These plasmids include isolated origins of replication (ori) of plasmids obtained from Acidithiobacillus ferrooxidans, represented by Sequence No 1 or by its reverse complementary sequence, and from Acidithiobacillus thiooxidans, represented by Sequence No 2 or by its reverse complementary Sequence. And Pnit, the expression promoter of reductase, isolated from the Wenelen (DSM 16786) strain, the property of Biosigma, represented by Sequence No 3. The advantage of the Pnit promoter, as will be described later, (FIG. 3), is that it strongly stimulates the expression of the gene it regulates.
Shuttle-type cloning genetic-vectors (plasmids) which include at least one of the ori represented in Sequence No 1 and Sequence No 2, expression promoter Pnit, Sequence No 3, a second origin of replication specific for another gene, a multiple cloning site, and at least one marker or reporter gene, are described.
The transformation method includes gradually modifying the conditions under which Acidithiobacillus spp. are cultured, in order to make them appropriate for being transformed with the plasmids of the invention.
Acidithiobacillus spp. cultures adapt to a pH of 5 to 7, and the substrate is modified into a tetrathionate salt. If necessary, the substrate is first modified from iron sulfide to elemental sulfur as an intermediate substrate, and finally into the tetrathionate salt. Once the culture is stable in these conditions, it is transformed by any known transformation technique, especially by electroporation.